Oligosaccharides from enzymatic cleavage of fucoidan

ABSTRACT

A novel endo-fucoidan-lyase and a novel microorganism useful in the production of sugar compounds. Sugar compounds represented by the following general formula (1), wherein at least one of alcoholic hydroxyl group has been sulfated, or salts thereof: ##STR1## wherein Y represents hydrogen or a group represented by the following formula (2). ##STR2##

This application is a 371 of PCT/JP96/01080, filed Apr. 22, 1996.

TECHNICAL FIELD

This invention relates to sugar compounds useful in the field of studieson carbohydrates, a polysaccharide lyase useful in the production ofthese sugar compounds and a microorganism belonging to the genusFucoidanobacter useful in the production of the sugar compounds.

BACKGROUND ART

There has been reported that fucoidan, which is a sulfatedpolysaccharide contained in brown algae (Phaeophyta), has variousbiological activities including anticoagulant, lipemia-clearing,antitumor, cancerous metastasis-inhibitory and anti-AIDS virus infectioneffects. Thus fucoidan is highly useful as a medicine.

When it is intended to use fucoidan as such as a medicine, however,there arise problems in antigenicity, uniformity, anticoagulantactivity, etc., since fucoidan is a sulfated polysaccharide having anextremely large molecular weight. Accordingly, it is often needed todegrade fucoidan to a certain extent.

It has been therefore desired to clarify the structure of fucoidan andreveal the relation thereof with its biological activities. However,fucoidan is a high-molecular compound carrying many branched chains andvarious constituting sugars. Moreover, its sulfate groups are bonded tovarious positions. These characteristics make it highly difficult toanalyze the structure of fucoidan. To analyze the structure of apolysaccharide, there is known a method comprising treating thepolysaccharide with an enzyme capable of degrading the same andanalyzing the structures of the oligosaccharides thus formed. Howeverthere has been commercially available neither any fucoidan degradingenzyme giving products with known sugar chain structure nor one servingas the standard of a fucoidan oligosaccharide from among those reportedhitherto.

For these reasons, there have been required sugar compounds withidentified structures, a polysaccharide degrading enzyme useful in theproduction of these sugar compounds and a microorganism useful in theproduction of the sugar compounds.

The present invention aims at providing sugar compounds usable inanalyzing the structure of fucoidan, identifying enzymatically degradedproducts of fucoidan and detecting the biological activities thereof, anovel endo-fucoidan-lyase useful in studies on fucoidan such as theproduction of fucoidan oligosaccharides and a novel microorganism usefulfor producing the sugar compounds.

In short, the first invention of the present invention relates to sugarcompounds represented by the following general formula (1) or (2),wherein at least one alcoholic hydroxyl group has been sulfated, orsalts thereof: ##STR3## wherein X represents hydrogen or a grouprepresented by the following formula (3): ##STR4## Y represents hydrogenor a group represented by the following formula (4) or (5): ##STR5##provided that X and Y are not hydrogen at the same time; and Zrepresents hydrogen or a group represented by the following formula (6):##STR6##

Examples of the compounds represented by the above general formula (1)or (2) are those represented by the following general formulae (7) to(15): ##STR7##

The second invention of the present invention relates to anendo-fucoidan-lyase characterized by having the followingphysicochemical properties.

(I) Function: acting on fucoidan and thus liberating at least thecompounds represented by the above formulae (7) and (8).

(II) Optimum pH value: ranging from pH 6 to 10.

(III) Optimum temperature: ranging from 30 to 40° C.

The third invention of the present invention relates to a bacteriumbelonging to the genus Fucoidanobacter which is a novel microorganismuseful in the production of the sugar compounds of the first inventionof the present invention, having menaquinone in the electron transportchain and containing 60% of GC [mole percent guanine plus cytosinecontent (mol % G+C)].

In the formulae (1), (2), (4) to (15) and (17) to (25) given herein, "˜"means that mannose or galactose occurs as both of α- and β-anomers.

The present inventors have found out that the sugar compounds of thepresent invention can be obtained by treating fucoidan with the enzymeof the second invention of the present invention or the cell extract orculture supernatant of the bacterium of the third invention of thepresent invention, thus completing the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the elution pattern of the sugar compound (a) having beenpyridyl-(2)-aminated (PA-a) which is eluted from an L-column.

FIG. 2 shows the elution pattern of the sugar compound (b) having beenpyridyl-(2)-aminated (PA-b) which is eluted from an L-column.

FIG. 3 shows the elution pattern of the sugar compound (c) having beenpyridyl-(2)-aminated (PA-c) which is eluted from an L-column.

FIG. 4 shows the elution pattern of the sugar compound (d) having beenpyridyl-(2)-aminated (PA-d) which is eluted from an L-column.

FIG. 5 shows the elution pattern of the sugar compound (e) having beenpyridyl-(2)-aminated (PA-e) which is eluted from an L-column.

FIG. 6 shows the elution pattern of the sugar compound (f) having beenpyridyl-(2)-aminated (PA-f) which is eluted from an L-column.

FIG. 7 shows the elution pattern of the sugar compound (g) having beenpyridyl-(2)-aminated (PA-g) which is eluted from an L-column.

FIG. 8 shows the elution pattern of the sugar compound (h) having beenpyridyl-(2)-aminated (PA-h) which is eluted from an L-column.

FIG. 9 shows the elution pattern of the sugar compound (i) having beenpyridyl-(2)-aminated (PA-i) which is eluted from an L-column.

FIG. 10 is the mass spectrogram (negative measurement) of the sugarcompound (a).

FIG. 11 is the mass spectrogram (negative measurement) of the sugarcompound (b).

FIG. 12 is the mass spectrogram (negative measurement) of the sugarcompound (c).

FIG. 13 is the mass spectrogram (negative measurement) of the sugarcompound (d).

FIG. 14 is the mass spectrogram (negative measurement) of the sugarcompound (e).

FIG. 15 is the mass spectrogram (negative measurement) of the sugarcompound (f).

FIG. 16 is the mass spectrogram (negative measurement) of the sugarcompound (g).

FIG. 17 is the mass spectrogram (negative measurement) of the sugarcompound (h).

FIG. 18 is the mass spectrogram (negative measurement) of the sugarcompound (i).

FIG. 19 is the mass-mass spectrogram (negative measurement) of the sugarcompound (a).

FIG. 20 is the mass-mass spectrogram (negative measurement) of the sugarcompound (b).

FIG. 21 is the mass-mass spectrogram (negative measurement) of the sugarcompound (c).

FIG. 22 is the mass-mass spectrogram (negative measurement) of the sugarcompound (d).

FIG. 23 is the mass-mass spectrogram (negative measurement) of the sugarcompound (e).

FIG. 24 is the mass-mass spectrogram (negative measurement) of the sugarcompound (f).

FIG. 25 is the mass-mass spectrogram (negative measurement) of the sugarcompound (g).

FIG. 26 is the mass-mass spectrogram (negative measurement) of the sugarcompound (h).

FIG. 27 is the mass-mass spectrogram (negative measurement) of the sugarcompound (i).

FIG. 28 is the ¹ H-NMR spectrum of the sugar compound (a).

FIG. 29 is the ¹ H-NMR spectrum of the sugar compound (b).

FIG. 30 is the ¹ H-NMR spectrum of the sugar compound (c).

FIG. 31 is the ¹ H-NMR spectrum of the sugar compound (d).

FIG. 32 is the ¹ H-NMR spectrum of the sugar compound (e).

FIG. 33 is the ¹ H-NMR spectrum of the sugar compound (f).

FIG. 34 is the ¹ H-NMR spectrum of the sugar compound (g).

FIG. 35 is the ¹ H-NMR spectrum of the sugar compound (h).

FIG. 36 is the ¹ H-NMR spectrum of the sugar compound (i).

FIG. 37 is a graph which shows the relationship between the relativeactivity (%) of the enzyme according to the second invention of thepresent invention and the pH value.

FIG. 38 is a graph which shows the relationship between the relativeactivity (%) of the enzyme according to the second invention of thepresent invention and the temperature (° C.).

FIG. 39 is a graph which shows the relationship between the residualactivity (%) of the enzyme according to the second invention of thepresent invention and the pH value at the treatment.

FIG. 40 is a graph which shows the relationship between the residualactivity (%) of the enzyme according to the second invention of thepresent invention and the temperature (° C.) at the treatment.

FIG. 41 shows the elution patterns of the sugar compounds (a) to (i)isolated by DEAE-Sepharose FF.

FIG. 42 shows the elution patterns of the sugar compounds (h) and (i)isolated by DEAE-Sepharose FF.

Now, the present invention will be described in greater detail.

The strain to be used in the second invention of the present inventionmay be an arbitrary one, so long as it belongs to the genusFlavobacterium and is capable of producing the endo-fucoidan-lyase ofthe present invention. As a particular example of the strain capable ofproducing the endo-fucoidan-lyase, citation can be made ofFlavobacterium sp. SA-0082 strain. The sugar compounds of the firstinvention of the present invention can be obtained by treating fucoidanwith the endo-fucoidan-lyase originating in this strain.

This strain, which has been found out for the first time by the presentinventors from seawater in Aomori, has the following mycologicalproperties.

1. Flavobacterium sp. SA-0082 Strain

a. Morphological Properties

    ______________________________________                                        (1)       Short rod;                                                                                      width: 0.8                                                                  -1.0 μm                                             length: 1.0-1.2 μm                                                        (2) Spore: none                                                               (3) Gram-staining: --                                                       ______________________________________                                    

b. Physiological Properties

(1) Growth temperature range: capable of growing at 37° C. or less,appropriate growth temperature ranging from 15 to 28° C.

    ______________________________________                                         (2)   Attitude to oxygen:                                                                              aerobic                                                (3) Catalase: +                                                               (4) Oxidase: +                                                                (5) Urease: weakly +                                                          (6) Acid formation D-glucose: +                                                lactose: +                                                                    maltose: +                                                                    D-mannitol: +                                                                 sucrose: -                                                                    trehalose: -                                                                 (7) Hydrolysis starch: -                                                       gelatin: +                                                                    casein: -                                                                     esculin: +                                                                   (8) Reduction of nitrate: -                                                   (9) Indole formation: -                                                      (10) Hydrogen sulfide formation: -                                            (11) Solidification of milk: -                                                (12) Sodium requirement: +                                                    (13) Salt requirement                                                          Growth in NaCl-free medium: -                                                 Growth in 1% NaCl medium: -                                                   Growth in seawater medium: +                                                 (14) Quinone: menaquinone 6                                                   (15) GC content of intracellular DNA: 32%                                     (16) OF-test: 0                                                               (17) Colony color: yellow                                                     (18) Motility: none                                                           (19) Gliding: none.                                                         ______________________________________                                    

It may be estimated that this strain is a bacterium analogous toFlavobacterium aquatile and Flavobacterium meningosepticum described inBergey's Manual of Systematic Bacteriology, Vol. 1 (1984) and Bergey'sManual of Determinative Bacteriology, Vol. 9 (1994). However, thisstrain differs from the former in incapable of forming any acid via themetabolism of sucrose, incapable of decomposing casein, capable ofdecomposing esculin, capable of liquefying gelatin and being positive tourease, and from the latter in incapable of decomposing casein andslowly growing at 37° C. Accordingly, this strain has been identified asa bacterium belonging to the genus Flavobacterium and namedFlavobacterium sp. SA-0082.

The above strain is designated as Flavobacterium sp. SA-0082 and hasbeen deposited at National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology (address: 1-3, Higashi1-chome, Tsukuba, Ibaragi, 305 JAPAN) under the accession number FERMP-14872 since Mar. 29, 1995 and deposited at National Institute ofBioscience and Human-Technology as described above under the accessionnumber FERM BP-5402 (transfer to international deposition was requestedon Feb. 15, 1996).

The nutrients to be added to the medium for incubating the strain to beused in the second invention of the present invention may be arbitraryones so long as the strain employed can utilize them so as to producethe endo-fucoidan-lyase of the second invention of the presentinvention. Appropriate examples of the carbon source include fucoidan,marine alga powder, alginic acid, fucose, glucose, mannitol, glycerol,saccharose, maltose, lactose and starch, while appropriate examples ofthe nitrogen source include yeast extract, peptone, casamino acids, cornsteep liquor, meat extract, defatted soybean, ammonium sulfate andammonium chloride. The medium may further contain inorganic substancesand metal salts such as sodium salts, phosphates, potassium salts,magnesium salts and zinc salts.

This strain also grows well in seawater or artificial seawatercontaining the above-mentioned nutrients.

In the incubation of the strain producing the endo-fucoidan-lyase of thesecond invention of the present invention, the yield of the enzymevaries depending on the incubation conditions. In general, it ispreferable that the incubation temperature ranges from 15 to 30° C. andthe pH value of the medium ranges from 5 to 9. The yield of theendo-fucoidan-lyase attains the maximum by incubating the strain underaeration and agitation for 5 to 72 hours. As a matter of course, theincubation conditions are appropriately selected depending on the strainemployed, the medium composition, etc. so as to achieve the maximumyield.

The endo-fucoidan-lyase of the second invention of the present inventionis contained in both of the cells and the culture supernatant.

The above-mentioned Flavobacterium sp. SA-0082 is incubated in anappropriate medium and the cells are harvested and disrupted by a meanscommonly employed for disrupting cells such as ultrasonication. Thus acell-free extract can be obtained.

Subsequently, the extract is purified by purification means commonlyemployed in the art to thereby give a purified enzyme preparation. Forexample, the purification may be effected by salting out, ion exchangechromatography, hydrophobic bond column chromatography, gel filtrationor the like to thereby give the purified endo-fucoidan-lyase of thesecond invention of the present invention free from any other fucoidandegrading enzymes.

The culture supernatant obtained by eliminating the cells from theabove-mentioned culture medium also contains a large amount of thisenzyme (extracellular enzyme) which can be purified by the same means asthose employed for purifying the intracellular enzyme.

The endo-fucoidan-lyase of the second invention of the present inventionhas the following chemical and physicochemical properties. Theextracellular enzyme is identical with the intracellular enzyme in theproperties other than molecular weight.

(I) Function: acting on fucoidan to cause the liberation of at least thesugar compounds represented by the above formulae (7) and (8).

(II) Optimum pH value: ranging from pH 6 to 10 (FIG. 37).

Namely, FIG. 37 is a graph which shows the relationship between therelative activity of this enzyme and the pH value wherein the ordinaterefers to the relative activity (%) while the abscissa refers to the pHvalue.

(III) Optimum temperature: ranging from 30 to 40° C. (FIG. 38).

Namely, FIG. 38 is a graph which shows the relationship between therelative activity of this enzyme and the temperature wherein theordinate refers to the relative activity (%) while the abscissa refersto the temperature (° C.).

(IV) pH stability: being stable within a range of from pH 6 to 11.5(FIG. 39).

Namely, FIG. 39 is a graph which shows the relationship between theresidual activity of this enzyme and the pH value at the treatmentwherein the ordinate refers to the residual activity (%) while theabscissa refers to the pH value.

(V) Temperature stability: being stable at temperatures of about 30° C.or less (FIG. 40).

Namely, FIG. 40 is a graph which shows the relationship between theresidual activity of this enzyme and the temperature at the treatmentwherein the ordinate refers to the residual activity (%) while theabscissa refers to the temperature (° C.).

(VI) Molecular weight: the molecular weight of this enzyme determined bygel filtration with the use of Sephacryl S-200 (mfd. by Pharmacia) isabout 70,000 in the case of the extracellular enzyme of the strainFlavobacterium sp. SA-0082 or about 460,000 in the case of theintracellular enzyme of this strain.

(VII) Method for measuring enzymatic activity:

The activity of the endo-fucoidan-lyase of the second invention of thepresent invention is measured in the following manner.

50 μl of a 2.5% solution of fucoidan originating in Kjellmaniellacrassifolia, 10 μl of the endo-fucoidan-lyase of the second invention ofthe present invention and 60 μl of an 83 mM phosphate buffer (pH 7.5)containing 667 mM of sodium chloride are mixed together and reacted at37° C. for 3 hours. Then 105 μl of the reaction mixture is mixed with 2ml of water under stirring and the absorbance (AT) is measured at 230nm. As controls, use is made of a reaction mixture prepared by the samemethod but substituting the endo-fucoidan-lyase of the second inventionof the present invention by the above-mentioned buffer alone employedfor dissolving the enzyme and another reaction mixture prepared by thesame method but substituting the fucoidan solution by water alone andthe absorbances (AB1 and AB2) thereof are also measured.

The amount of the enzyme by which 1 μmol of the glycoside bond betweenmannose and uronic acid can be exclusively cleaved in one minute isreferred to as one U. The bond thus cleaved is determined by taking themillimolar molecular extinction coefficient of the unsaturated uronicacid formed in the elimination reaction as 5.5. The activity of theenzyme is determined in accordance with the following equation:

    Activity (U/ml)=(AT-AB1-AB2)×2.105×120/(5.5×105×0.01×180);

2.105: volume (ml) of the sample the absorbance of which is to bemeasured;

120: volume (μl) of the enzyme reaction mixture;

5.5: millimolar molecular extinction coefficient (/mM) of unsaturateduronic acid at 230 nm;

105: volume (μl) ofthe reaction mixture employed for dilution;

0.01: volume (ml) of the enzyme; and

180: reaction time (min).

The protein is determined by measuring the absorbance of the enzymesolution at 280 nm and calculated by taking the absorbance of the 1mg/ml protein solution as 1.0.

The present inventors have determined the action mechanism of theendo-fucoidan-lyase of the second invention of the present invention inthe following manner.

(1) Preparation of Kjellmaniella crassifolia Fucoidan

Dry Kjellmaniella crassifolia is ground with a free mill Model M-2 (mfd.by Nara Kikai Seisakusho) and treated in 10 times as much 85% methanolat 70° C. for 2 hours. Then it is filtered and the residue is furthertreated in 10 times as much methanol at 70° C. for 2 hours. Afterfiltering, 20 times as much water is added to the residue. Then themixture is treated at 100° C. for 3 hours and filtered to thereby givean extract. The salt concentration of the extract is adjusted to thesame level as that of a 400 mM solution of sodium chloride. Thencetylpyridinium chloride is added thereto until no precipitate is formedany more. After centrifuging, the precipitate is thoroughly washed withethanol to thereby completely eliminate the cetylpyridinium chloride.Next, it is subjected to desalting and the removal of low-molecularweight substances by using an ultrafilter (exclusion molecular weight ofultrafiltration membrane: 100,000, mfd. by Amicon). The precipitate thusformed is eliminated by centrifugation. The supernatant is freeze-driedto thereby give purified Kjellmaniella crassifolia fucoidan. The yieldof the product is about 4% based on the weight of the dry Kjellmaniellacrassifolia powder.

(2) Degradation of Fucoidan by endo-fucoidan-lyase and Purification ofDegradation Product

The purified fucoidan originating in Kjellmaniella crassifolia istreated with the endo-fucoidan-lyase of the second invention of thepresent invention to thereby give the degradation products in largeamounts.

Namely, 600 ml of a 5% solution of the fucoidan originating inKjellmaniella crassifolia, 750 ml of a 100 mM phosphate buffer (pH 8.0),150 ml of 4 M sodium chloride and 3.43 ml of a 1750 mU/ml solution ofthe endo-fucoidan-lyase of the present invention are mixed together andreacted at 25° C. for 144 hours.

Then the reaction mixture is dialyzed by using a dialysis membrane of3500 in pore size and a fraction of molecular weight of 3500 or less istaken up. After desalting with a Micro Acilyzer G3 (mfd. by AsahiChemical Industry Co., Ltd.), this fraction is fractionated into ninefractions (a), (b), (c), (d), (e), (f), (g), (h) and (i) withDEAE-Sepharose FF. FIG. 41 shows the elution patterns thereof whereinthe abscissa refers to the fraction number, the left ordinate and closecircles refer to the sugar content of the sample determined by thephenol-sulfuric acid method and expressed in the absorbance at 480 nm,the right ordinate and open circles refer to the unsaturated glucuronicacid content of the sample expressed in the absorbance at 235 nm, andthe rightmost ordinate and the dotted line refer to the ammonium acetateconcentration (M) in the eluate. The fraction numbers of the fractionsare respectively as follows: (a): from 42 to 43, (b): from 84 to 91,(c), from 51 to 52, (d) 79, (e): from 102 to 103, (f): from 62 to 63,(g): 45, (h): 75, and (i): 77.

With respect to the fractions (h) and (i), the above-mentioned fractionsof Nos. 64 to 78 are combined and re-purified with DEAE-Sepharose FF.FIG. 42 shows the elution patterns thereof wherein the abscissa refersto the fraction number, the left ordinate and close circles refer to thesugar content of the sample determined by the phenol-sulfuric acidmethod and expressed in the absorbance at 480 nm, the right ordinate andopen circles refer to the unsaturated glucuronic acid content of thesample expressed in the absorbance at 235 nm, and the rightmost ordinateand the dotted line refer to the ammonium acetate concentration (M) inthe eluate. The fraction numbers of the fractions are respectively asfollows: (h): from 92 to 96, and (i): from 99 to 103.

(3) Analysis of the Structure of Enzymatic Reaction Product

1 Confirmation of Uniformity of Each Fraction

A portion of each of the above-mentioned nine fractions (a), (b), (c),(d), (e), (f), (g), (h) and (i) is pyridyl-(2)-aminated (PA) at thereducing end by using GlycoTAG and GlycoTAG Reagent Kit (each mfd. byTakara Shuzo Co., Ltd.) to thereby give PA saccharides (PA-a), (PA-b),(PA-c) (PA-d), (PA-e), (PA-f), (PA-g), (PA-h) and (PA-i), which are thenanalyzed by HPLC. Thus it is confirmed that (PA-a), (PA-b), (PA-c)(PA-d), (PA-e), (PA-f), (PA-g), (PA-h) and (PA-i) are each a uniformsubstance.

The HPLC is effected under the following conditions.

Apparatus: Model L-6200 (mfd. by Hitachi, Ltd.).

Column: L-column (4.6×250 mm) [Kagaku Yakuhin Kensa Kyokai(Foundation)].

Eluent:

50 mM acetic acid-triethylamine (pH 5.5) for the substances of the aboveformulae (7), (8) and (9) [i.e., (PA-a), (PA-b) and (PA-c)];

100 mM acetic acid-triethylamine (pH 5) for the substances of the aboveformulae (10), (12) (13) and (15) [i.e., (PA-d), (PA-f), (PA-g) and(PA-i)];

200 mM acetic acid-triethylamine (pH 3.8) from 0 to 10 minutes and 200mM acetic acid-triethylamine (pH 3.8) and 200 mM aceticacid-triethylamine (pH 3.8) containing 0.5% of tetrahydrofuran from 10to 60 minutes while linearly changing the ratio of the former to thelatter from 100:0 to 20:80 for the substance of the above formula (11)[i.e., (PA-e)]; and 200 mM acetic acid-triethylamine (pH 3.8) and 200 mMacetic acid-triethylamine (pH 3.8) containing 0.5% of tetrahydrofranfrom 0 to 60 minutes while linearly changing the ratio of the former tothe latter from 80:20 to 50:50 for the substance of the above formula(14) [i.e., (PA-h)].

Detection: Fluorometric Detector F-1150 (mfd. by Hitachi, Ltd.),excitation wavelength: 320 nm, fluorescent wavelength: 400 nm.

Flow rate: 1 ml/min.

Column temperature: 40° C.

2 Analysis of Reducing End Sugar and Neutral Sugar Composition ofEnzymatic Reaction Product

The PA-sugars (PA-a), (PA-b), (PA-c), (PA-d), (PA-e), (PA-f), (PA-g),(PA-h) and (PA-i) are each hydrolyzed by treating with 4 N hydrochloricacid at 100° C. for 3 hours and the reducing end sugar is examined byHPLC.

Subsequently, the reducing ends of these hydrolyzates arepyridyl-(2)-aminated (PA) by using GlycoTAG and GlycoTAG Reagent Kit(each mfd. by Takara Shuzo Co., Ltd.) and the neutral sugar compositionsare analyzed by HPLC. The HPLC is effected under the followingconditions.

Apparatus: Model L-6200 (mfd. by Hitachi, Ltd.).

Column: PALPAK Type A (4.6 mm×150 mm, mfd. by Takara Shuzo, Co., Ltd.).

Eluent: 700 mM borate buffer (pH 9.0) acetonitrile=9:1.

Detection: Fluorometric Detector F-1150 (mfd. by Hitachi, Ltd.),excitation wavelength: 310 nm, fluorescent wavelength: 380 nm;

Flow rate: 0.3 ml/min; and

Column temperature: 65° C.

As a result, it is found out that (PA-a), (PA-b), (PA-c), (PA-d),(PA-e), (PA-f) and (PA-i) all carry mannose as the reducing end sugar.Regarding the neutral sugarcomposition, (PA-a), (PA-b), (PA-c), (PA-e),(PA-f) and (PA-i) contain mannose and fucose in equimolar amounts while(PA-d) contains mannose and fucose at a ratio of 2:1.

(PA-g) and (PA-h) each carry galactose as the reducing end sugar.Regarding the neutral sugar composition, (PA-g) contains galactose andfucose at a ratio of 1:2 while (PA-h) contains galactose and fucose at aratio of 2:1.

Further, the configuration of mannose which is one of the constitutingsugars is examined in the following manner. By using F-KitsGlucose/Fructose and Phosphomannose Isomerase (each mfd. by BoehringerMannheim-Yamanouchi), a reaction system by which D-mannose alone can bedetermined is constructed in accordance with the manufacturer'sdescription. Separately, 100 μg portions of the sugar compounds (a),(b), (c), (d), (e), (f) and (i) are each hydrolyzed with 2 Nhydrochloric acid at 100° C. for 3 hours and, after neutralization,subjected to the determination in this reaction system. As a result,D-mannose is detected from all of the sugar compounds (a), (b), (c),(d), (e), (f) and (i). Thus it is proved that the sugar compounds (a),(b), (c), (d), (e), (f) and (i) all have D-mannose as the constitutingsugar.

Further, the configuration of galactose which is one of the constitutingsugars of (g) and (h) is examined by using F-Kit Lactose/Galactose (mfd.by Boehringer Mannheim-Yamanouchi) by which D-galactose alone can bedetected. Namely, 100 μg portions of (g) and (h) are each hydrolyzedwith 2 N hydrochloric acid at 100° C. for 3 hours and, afterneutralization, subjected to the determination in this reaction system.As a result, galactose is detected from (g) and (h). Thus it is provedthat (g) and (h) both have D-galactose as the constituting sugar.

Furthermore, the configuration of fucose which is another constitutingsugar is examined in the following manner. In accordance with the methoddescribed in Clinical Chemistry, 36, 474-476 (1990), 100 μg portions ofthe above-mentioned compounds (a), (b), (c), (d), (e), (f), (g), (h) and(i) are hydrolyzed with 2 N hydrochloric acid at 100° C. for 3 hoursand, after neutralization, subjected to the determination in thisreaction system by which not D-fucose but L-fucose alone can bedetected. As a result, L-fucose is detected from the sugar compounds(a), (b), (c), (d), (e), (f), (g), (h) and (i).

3 Analysis of Molecular Weight of Enzymatic Reaction Product

Next, the sugar compounds (a), (b), (c), (d), (e), (f), (g), (h) and (i)are subjected to mass spectrometry with the use of an API-III massspectrograph (mfd. by Perkin-Elmer Science). Thus, it is found out thatthese substances have molecular weights of 564, 724, 1128, 1062, 1448,644, 632, 1358 and 1288, respectively. In (b) and (c), divalent anionsform major signals. A monovalent ion, a monovalent ion having sodiumattached thereto and a divalent ion are detected from (d). A divalention having four sodium ions attached thereto, a trivalent ion havingthree sodium ions attached thereto, a tetravalent ion having a sodiumion attached thereto, etc. are detected from (e). A monovalent ionhaving two sodium ions attached thereto is detected from (f). Amonovalent ion, a monovalent ion having sodium attached thereto and adivalent ion are detected from (g). Monovalent, divalent, trivalent andtetravalent ions respectively having four, three, two and one sodiumion, etc. are detected from (h). A monovalent ion from which two sulfategroups have been eliminated and to which a sodium ion has been attachedand a divalent ion from which two sulfate groups have been eliminatedare detected from (i).

By the mass-mass (MS/MS) spectrometry of the negative mode, detection ismade from (a) of a monovalent sulfate ion (molecular weight 97), amonovalent ion (molecular weight 157) wherein a water molecule and ahydrogen ion have been eliminated from an unsaturated hexuronic acid, amonovalent ion (molecular weight 175) wherein a hydrogen ion has beeneliminated from an unsaturated hexuronic acid, a monovalent ion(molecular weight 225) wherein a water molecule and a hydrogen ion havebeen eliminated from sulfated fucose, a monovalent ion (molecular weight243) wherein a hydrogen ion has been eliminated from sulfated fucose, amonovalent ion (molecular weight 319) wherein a water molecule and ahydrogen ion have been eliminated from an unsaturated hexuronic acidbonded to mannose, and a monovalent ion (molecular weight 405) wherein ahydrogen ion has been eliminated from sulfated fucose bonded to mannose.

By the MS/MS spectrometry of the negative mode, detection is madesimilarly from (b) of a monovalent sulfate ion (molecular weight 97), amonovalent ion (molecular weight 175) wherein a hydrogen ion has beeneliminated from an unsaturated hexuronic acid, a monovalent ion(molecular weight 243) wherein a hydrogen ion has been eliminated fromsulfated fucose, a divalent ion (molecular weight 321) wherein twohydrogen ions have been eliminated from an unsaturated hexuronic acidand sulfated fucose bonded to sulfated mannose, a monovalent ion(molecular weight 405) wherein a hydrogen ion has been eliminated fromsulfated fucose bonded to mannose or fucose bonded to sulfated mannose,and a monovalent ion (molecular weight 417) wherein a hydrogen ion hasbeen eliminated from an unsaturated hexuronic acid bonded to sulfatedmannose.

By the MS/MS spectrometry of the negative mode, detection is made from(c) of a monovalent sulfate ion (molecular weight 97), a monovalent ion(molecular weight 175) wherein a hydrogen ion has been eliminated froman unsaturated hexuronic acid, a monovalent ion (molecular weight 225)wherein a water molecule and a hydrogen ion have been eliminated fromsulfated fucose, a monovalent ion (molecular weight 243) wherein ahydrogen ion has been eliminated from sulfated fucose, a divalent ion(molecular weight 371) wherein two hydrogen ions have been eliminatedfrom sulfated fucose bonded to mannose bonded to a hexuronic acid bondedto mannose, a monovalent ion (molecular weight 405) wherein a hydrogenion has been eliminated from sulfated fucose bonded to mannose, and amonovalent ion (molecular weight 721) wherein water and a hydrogen ionhave been eliminated from sulfated fucose and an unsaturated hexuronicacid bonded mannose bonded to hexuronic acid.

By the MS/MS spectrometry of the negative mode, detection is made fromthe divalent ions of (d) of a monovalent sulfate ion (molecular weight97), a monovalent ion (molecular weight 175) wherein a hydrogen ion hasbeen eliminated from an unsaturated hexuronic acid, a monovalent ion(molecular weight 225) wherein a water molecule and a hydrogen ion havebeen eliminated from sulfated fucose, a monovalent ion (molecular weight243) wherein a hydrogen ion has been eliminated from sulfated fucose, amonovalent ion (molecular weight 405) wherein a hydrogen ion has beeneliminated from sulfated fucose bonded to mannose, a divalent ion(molecular weight 450) wherein two sulfate groups and two hydrogen ionshave been eliminated from (d), and a divalent ion (molecular weight 490)wherein sulfate group and two hydrogen ions have been eliminated from(d).

By the MS/MS spectrometry of the negative mode, detection is made from(e) of a monovalent sulfate ion (molecular weight 97), a monovalent ion(molecular weight 225) wherein a water molecule and a hydrogen ion havebeen eliminated from sulfated fucose, a monovalent ion (molecular weight243) wherein a hydrogen ion has been eliminated from sulfated fucose, amonovalent ion (molecular weight 345) wherein two hydrogen ions havebeen eliminated from and a sodium ion has been attached to disulfatedfucose, a trivalent ion (molecular weight 450) wherein two sulfategroups have been eliminated, three sodium ions have been attached andsix hydrogen ions have been eliminated from (e), a trivalent ion(molecular weight 476) wherein a sulfate group and six hydrogen ionshave been eliminated from (e) and three sodium ions have been attachedthereto, a monovalent ion (molecular weight 563) wherein a hydrogen ionhas been eliminated from an unsaturated hexuronic acid and sulfatedfucose bonded to mannose, and a monovalent ion (molecular weight 705)wherein a water molecule and a hydrogen ion have been eliminated from anunsaturated hexuronic acid and disulfated fucose bonded to sulfatedmannose.

By the MS/MS spectrometry of the negative mode, detection is made from(f) of a monovalent sulfate ion (molecular weight 97), a monovalent ion(molecular weight 243) wherein a hydrogen ion has been eliminated fromsulfated fucose, and a monovalent ion (molecular weight 421) whereinwater and two hydrogen ions have been eliminated from and a sodium ionhas been attached to an unsaturated hexuronic acid bonded to sulfatedmannose.

By the MS/MS spectrometry of the negative mode, detection is made from(g) of a monovalent ion (molecular weight 405) wherein a hydrogen ionhas been eliminated from sulfated fucose bonded to galactose and amonovalent ion (molecular weight 551) wherein a hydrogen ion has beeneliminated from sulfated fucose bonded to fucose bonded to galactose orfucose bonded to sulfated fucose bonded to galactose.

By the MS/MS spectrometry of the negative mode, detection is made fromthe substance, wherein three sodium ions have been attached to (h) andfive hydrogen ions have been eliminated therefrom, of a monovalentsulfate ion (molecular weight 97), a monovalent ion (molecular weight225) wherein a water molecule and a hydrogen ion have been eliminatedfrom sulfated fucose, and a monovalent ion (molecular weight 1197)wherein a hydrogen ion and two sulfate groups have been eliminated from(h).

By the MS/MS spectrometry of the negative mode, detection is made fromthe divalent ion, wherein two sulfate groups and two hydrogen ions havebeen eliminated from (i), of a monovalent sulfate ion (molecular weight97) and a monovalent ion (molecular weight 345) wherein two hydrogenions have been eliminated from and a sodium ion has been attached todisulfated fucose.

4 Analysis of Sugar Composition of Enzymatic Reaction Product

As the above results of mass spectrometry indicate, there is a highpossibility that the sugar compounds (a), (b), (c), (d), (e), (f) and(i) might each contain an unsaturated hexuronic acid in its molecule.

Thus the following experiment is conducted to prove that these enzymaticreaction products each contains a hexuronic acid carrying an unsaturatedbond in its molecule. It is known that a strong absorption at 230 to 240nm is assignable to an unsaturated bond in a molecule. Thus, theabsorbance of the aqueous solution of each of the purifiedoligosaccharides (a), (b), (c), (d), (e), (f), (g), (h) and (i) ismeasured at 230 to 240 nm. As a result, the aqueous solutions of (a),(b), (c), (d), (e), (f) and (i) each shows a strong absorption, whichsuggests the presence of an unsaturated bond in the molecule. It is alsoconfirmed that the absorbance at 230 to 240 nm increases as thedegradation of fucoidan by this enzyme proceeds. These facts stronglysuggest that this enzyme would cleave the glycoside bond between mannoseand a hexuronic acid or galactose and a hexuronic acid in fucoidan viaan elimination reaction.

Most of the enzymatic reaction products have an unsaturated hexuronicacid at the nonreducing ends and carry mannose at the reducing ends,which suggests that the fucoidan prepared involves a molecular speciescomposed of a hexuronic acid and mannose alternately bonded to eachother.

Because of containing fucose as the main constituting saccharide,fucoidan is more liable to be degraded with acids than commonpolysaccharides. On the other hand, it is known that the bonds ofhexuronic acids and mannose are relatively highly tolerant to acids. Thepresent inventors have attempted to identify the hexuronic acid in thesugar chain which is composed of the hexuronic acid and mannosealternately bonded to each other and contained in the fucoidanoriginating in Kjellmaniella crassifolia in the following manner withreference to the method described in Carbohydrate Research, 125, 283-290(1984). First, the fucoidan is dissolved in 0.3 M oxalic acid andtreated at 100° C. for 3 hours. Then it is subjected to molecular weightfractionation and fractions of molecular weight of 3,000 or more arecombined. Then it is further treated with an anion exchange resin andthe adsorbed matters are collected. The substance thus obtained isfreeze-dried and hydrolyzed with 4 N hydrochloric acid. After adjustingthe pH value to 8, it is pyridyl-(2)-aminated and uronic acid isanalyzed by HPLC. The HPLC is effected under the following conditions.

Apparatus: Model L-6200 (mfd. by Hitachi, Ltd.);

Column: PALPAK Type N (4.6 mm×250 mm, mfd. by Takara Shuzo, Co., Ltd.);

Eluent: 200 mM acetic acid-triethylamine buffer (pH 7.3): acetonitrile25:75;

Detection: Fluorometric Detector F-1150 (mfd. by Hitachi, Ltd.),excitation wavelength: 320 nm, fluorescent wavelength: 400 nm;

Flow rate: 0.8 ml/min;

Column temperature: 40° C.

As the standards for PA hexuronic acids, use is made of those preparedby pyridyl-(2)-amination of glucuronic acid manufactured by SigmaChemical Co., galacturonic acid manufactured by Wako Pure ChemicalIndustries, Ltd., iduronic acid obtained by hydrolyzing4-methylumbelliferyl-α-L-iduronide manufactured by Sigma Chemical Co.,and mannuronic acid and guluronic acid obtained by hydrolyzing alginicacid (mfd. by Wako Pure Chemical Industries, Ltd.) in accordance withthe method described in Acta Chemica Scandinavicaj, 15, 1397-1398 (1961)followed by the separation with an anion exchange resin.

As a result, it is found out that glucuronic acid alone is contained asthe hexuronic acid in the above-mentioned molecular species of fucoidan.

Further, the glucuronic acid in the hydrolyzate of the above-mentionedmolecular species is separated from D-mannose by using an anion exchangeresin and freeze-dried. Then the specific rotation thereof is measured.It is thus clarified that the glucuronic acid is a dextrorotatory one,i.e., D-glucuronic acid.

Further, the fucoidan originating in Kjellmaniella crassifolia istreated with the endo-fucoidan hydrolase of the second invention of thepresent invention and then hydrolyzed with the use of oxalic acidsimilar to the above case. However, no polymer having D-glucuronic acidand D-mannose alternately bonded to each other is found out.

Based on these results, it is clarified that the enzyme of the presentinvention cleaves, via the elimination reaction, a molecular specieshaving a skeleton structure composed of D-glucuronic acid and D-mannosealternately bonded to each other.

Further, the polymer obtained by the hydrolysis with oxalic acid issubjected to NMR spectrometry to thereby examine the anomericconfiguration of the binding sites of D-glucuronic acid and D-mannoseand the glycoside bond.

The obtained NMR spectra of the polymer are as follows. The chemicalshifts in the ¹ H-NMR spectra are expressed by taking the chemical shiftof the methyl group in triethylamine as 1.13 ppm, while those in the ¹³C-NMR spectra are expressed by taking the chemical shift of the methylgroup in triethylamine as 9.32 ppm.

¹ H-NMR(D₂ O); δ5.25(1H, br-s, 1-H), 4.32(1H, d, J=7.6 Hz, 1'-H),4.00(1H, br-s, 2-H), 3.71(1H, m, 5'-H), 3.69(1H, m, H of 5-CH), 3.68(1H,m, 3-H), 3.63(1H, m, H of 5-CH), 3.63(1H, m, 4'-H), 3.57(1H, m, 4-H),3.54(1H, m, 3'-H), 3.53(1H, m, 5-H), 3.25(1H, t, J=8.5 Hz, 2'-H); ¹³C-NMR(D₂ O); δ175.3(C of 5'-COOH), 102.5(1'-C), 99.6(1-C), 78.5(2-C),77.9(4'-C), 77.0(3'-C), 76.7(5'-C), 73.9(5-C), 73.7(2'-C), 70.6(3-C),67.4(4-C), 61.05(C of 5-CH₂ OH).

The peaks are assignable respectively to the positions shown by thenumerical values in the following formula (16): ##STR8##

Regarding the configuration at the 1-position of the D-glucuronic acid,it is identified as β-D-glucuronic acid because of its vicinal bindingconstant of 7.6 Hz.

Regarding the configuration at the 1-position of the D-mannose, it isidentified as α-D-mannose because of its chemical shift of 5.25 ppm.

The binding manners of the constituting sugars are analyzed by the HMBCmethod, i.e., the ¹ H-dtected multiple-bond heteronuclear multiplequantum coherence spectrum.

The DQF-COSY and HOHAHA methods are employed in the assignment in the ¹H-NMR spectra while the HSQC method is employed in the assignment in the¹³ C-NMR spectra.

In the HMBC spectrum, cross peaks are observed between 1-H and 4'-C withbetween 4'-H and 1-C, and between 1'-H and 2-C with between 2-H and1'-C. These facts indicate that D-glucuronic acid is bonded to the2-position of D-mannose via a β-bond while D-mannose is bonded to the4-position of D-glucuronic acid via an α-bond.

5 Analysis on Sugar Binding Manner and Binding Site of Sulfate Group inEnzymatic Reaction Product

To examine the binding manner of the constituting sugars and sulfategroups, the enzymatic reaction products are analyzed by NMR spectrometrywith the use of a nuclear magnetic resonance spectrometer Model JNM-α500(500 Mz; mfd. by JEOL Ltd.). The analytical data thus obtained indicatethat the sugar compounds (a), (b), (c), (d), (e), (f), (g), (h) and (i)are represented respectively by the above formulae (7), (8), (9), (10),(11), (12), (13), (14) and (15). That is tosay, the facts thus clarifiedare as follows. The sugar compound (a) has a structure whereinunsaturated D-glucuronic acid and L-fucose having a sulfate group bondedthereto are attached to D-mannose which is a reducing end residue. Thesugar compound (b) has a structure wherein unsaturated D-glucuronic acidand L-fucose having two sulfate groups bonded thereto are attached toD-mannose which is a reducing end residue having a sulfate group bondedthereto. The sugar compound (c) has a structure wherein D-glucuronicacid and L-fucose having a sulfate group bonded thereto are attached toD-mannose which is a reducing end residue, D-mannose is attached furtherto the D-glucuronic acid and unsaturated D-glucuronic acid, and L-fucosehaving a sulfate group bonded thereto are attached furthermore to theD-mannose. The sugar compound (d) has a structure wherein a sulfategroup, D-glucuronic acid and L-fucose having two sulfate groups bondedthereto are attached to D-mannose which is a reducing end residue,D-mannose is attached further to the D-glucuronic acid, and unsaturatedD-glucuronic acid is attached furthermore to the D-mannose. The sugarcompound (e) has a structure wherein a sulfate group, D-glucuronic acidand L-fucose having two sulfate groups bonded thereto are attached toD-mannose which is a reducing end residue, D-mannose having a sulfategroup bonded thereto is attached further to the D-glucuronic acid, andL-fucose having two sulfate groups bonded thereto and unsaturatedD-glucuronic acid are attached furthermore to the D-mannose. The sugarcompound (f) has a structure wherein unsaturated D-glucuronic acid andL-fucose having a sulfate group bonded thereto are attached to D-mannosewhich is a reducing end residue having a sulfate group bonded thereto.The sugar compound (g) has a structure wherein L-fucose having a sulfategroup bonded thereto is attached to D-galactose which is a reducing endresidue and L-fucose having a sulfate group is attached further to theL-fucose. The sugar compound (h) has a structure consisting of twobranched chains starting with D-galactose which is a reducing endresidue having a sulfate group bonded thereto wherein L-fucose having asulfate group bonded thereto is attached to D-galactose and L-fucosehaving a sulfate group is attached further to the L-fucose in one of thesugar chains while D-galactose having a sulfate group bonded thereto isattached to D-galactose having a sulfate group bonded thereto in anothersugar chain. The sugar compound (i) has a structure wherein a sulfategroup, D-glucuronic acid and L-fucose having a sulfate group bondedthereto are attached to D-mannose which is a reducing end residue,D-mannose is attached further to the D-glucuronic acid, and L-fucosehaving two sulfate groups bonded thereto and unsaturated D-glucuronicacid are attached furthermore to the D-mannose.

The compounds involved in the first invention of the present inventionare obtained by treating fucoidan with the endo-fucoidan-lyase of thesecond invention of the present invention.

Next, the physical properties of the compounds represented by theformulae (7), (8), (9), (10), (11), (12), (13), (14) and (15), i.e., thesugar compounds (a), (b), (c), (d), (e), (f), (g), (h) and (i) which areexamples of the sugar compounds of the present invention will bedescribed.

FIGS. 1, 2, 3, 4, 5, 6, 7, 8 and 9 show the HPLC elution patterns of thepyridyl-(2)-aminated sugar compounds (PA-a), (PA-b), (PA-c), (PA-d),(PA-e), (PA-f), (PA-g), (PA-h) and (PA-i), respectively. In each figure,the ordinate refers to the relative fluorescence intensity while theabscissa refers to the retention time (min).

Further, FIGS. 10, 11, 12, 13, 14, 15, 16, 17 and 18 show the massspectra of the sugar compounds (a), (b), (c), (d), (e), (f), (g), (h)and (i), respectively, while FIGS. 19, 20, 21, 22, 23, 24, 25, 26 and 27show the mass-mass spectra of the sugar compounds (a), (b), (c), (d),(e), (f), (g), (h) and (i), respectively. In each figure, the ordinaterefers to the relative intensity (%) while the abscissa refers to m/z.

Furthermore, FIGS. 28, 29, 30, 31, 32, 33, 34, 35 and 36 show the ¹H-NMR spectra of the sugar compounds (a), (b), (c), (d), (e), (f), (g),(h) and (i), respectively.

In each figure, the ordinate refers to the signal intensity while theabscissa refers to the chemical shift (ppm).

The chemical shifts in the ¹ H-NMR spectra are expressed by taking thechemical shift of HOD as 4.65 ppm.

Physical properties of the compound (a):

Molecular weight: 564.

MS m/z: 563 [M-H⁺ ]⁻.

MS/MSm/z: 97 (HSO₄ ]⁻, 157 [unsaturated D-glucuronic acid-H₂ O-H⁺ ]⁻,175 [unsaturated D-glucuronic acid-H⁺ ]⁻, 225 [sulfated L-fucose-H₂ O-H⁺]⁻, 243 [sulfated L-fucose-H⁺ ]⁻, 319 [unsaturated D-glucuronic acidbonded to D-mannose-H₂ O-H⁺ ]⁻, 405 [M-unsaturated D-glucuronic acid-H⁺]⁻, 483 [M-SO₃ -H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.78(1H, d, J=3.7 Hz, 4"-H), 5.26(1H, d, J=1.2 Hz, 1-H),5.12(1H, d, J=4.0 Hz, 1'-H), 5.03(1H, d, J=6.1 Hz, 1"-H), 4.47(1H, d-d,J=3.4, 10.4 Hz, 3'-H), 4.21(1H, br-s, 2-H), 4.12(1H, m, 5'-H), 4.10(1H,d-d, J=3.7, 5.8 Hz, 3"-H), 4.03(1H, d, J=3.4 Hz, 4'-H), 3.86(1H, m,3-H), 3.83(1H, d-d, J=4.0, 10.4 Hz, 2'-H), 3.72(1H, m, 4-H), 3.72(1H, m,5-H), 3.70(2H, m, H₂ of 5-CH₂), 3.65(1H, d-d, J=5.8, 6.1 Hz, 2"-H),1.08(3H, d, J=6.7 Hz, H₃ of 5'-CH₃).

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-mannose=1:1:1 (each onemolecule).

Sulfate Group:

one molecule (at the 3-position of L-fucose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (17):##STR9##

Physical properties of the compound (b):

Molecular weight: 724.

MS m/z: 723 [M-H⁺ ]⁻, 361 [M-2H⁺ ]²⁻.

MS/MS m/z: 97 [HSO₄ ]⁻, 175 [unsaturated D-glucuronic acid-H⁺ ]⁻, 243[sulfated L-fucose-H⁺ ]⁻, 321 [M-SO₃ -2H⁺ ]²⁻, 405 [M-unsaturatedD-glucuronic acid-2SO₃ -H⁺ ]⁻, 417 (M-L-fucose-2SO₃ -H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.66(1H, d, J=3.4 Hz, 4"-H), 5.27(1H, d, J=7.3 Hz,1"-H), 5.25(1H, d, J=1.8 Hz, 1-H), 5.21(1H, d, J=3.7 Hz, 1'-H), 4.50(1H,d, J=3.1 Hz, 4'-H), 4.32(1H, q, J=6.7 Hz, 5'-H), 4.27(1H, d-d, J=3.7,10.4 Hz, 2'-H), 4.21(1H, d-d, J=3.4, 6.7 Hz, 3"-H), 4.18(1H, d-d, J=1.8,11.0 Hz, H of 5-CH), 4.15(1H, br-s, 2-H), 4.10(1H, d-d, J=5.8, 11.0 Hz,H of 5-CH), 3.99(1H, d-d, J=3.1, 10.4 Hz, 3'-H), 3.90(1H, m, 5-H),3.82(1H, m, 3-H), 3.82(1H, m, 4-H), 3.54(1H, br-t, J=7.3 Hz, 2"-H),1.11(3H, d, J=6.7 Hz, H₃ of 5'-CH₃).

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-mannose=1:1:1 (each onemolecule).

Sulfate Group:

three molecules (at the 2- and 4-positions of L-fucose and the6-position of D-mannose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (18):##STR10##

Physical properties of the compound (c):

Molecular weight: 1128.

MS m/z: 1127 [M-H⁺ ]⁻.

MS/MS m/z: 97 [HSO₄ ]⁻, 175 [unsaturated D-hexuronicacid-H⁺ ]⁻, 225[sulfated L-fucose-H₂ O-H⁺ ]⁻, 243 [sulfated L-fucose-H⁺ ]⁻, 371[M-unsaturated D-glucuronic acid-L-fucose-SO₃ -2H⁺ ]²⁻, 405 [sulfatedL-fucose bonded to D-mannose-H⁺ ]⁻, 721 [M-D-mannose-L-fucose-SO₃ -H₂O-H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.69(1H, d, J=3.7 Hz, (4)"-H), 5.34(1H, s, (1)-H),5.16(1H, s, 1-H), 5.10(1H, d, J=4.0 Hz, (1)'-H), 5.05(1H, d, J=3.7 Hz,1'-H), 4.93(1H, d, J=6.4 Hz, (1)"-H), 4.50(1H, d-d, J=3.4, 10.7 Hz,3'-H), 4.47(1H, d-d, J=3.4, 10.4 Hz, (3)'-H), 4.39(1H, d, J=7.9 Hz,1"-H), 4.33(1H, br-s, (2)-H), 4.14(1H, m, 2-H), 4.12(1H, m, (3)"-H),4.12(1H, m, 5'-H), 4.12(1H, m, (5)'-H), 4.04(1H, m, 4'-H), 4.03(1H, m,(4)'-H), 3.85(1H, m, 2'-H), 3.85(1H, m, (2)'-H), 3.82(1H, m, 3-H),3.82(1H, m, (3)-H), 3.73(1H, m, 4-H), 3.73(1H, m, 5-H), 3.73(1H, m,(4)-H), 3.70(2H, m, H₂ of 5-CH₂), 3.70(2H, m, H₂ of (5)-CH₂), 3.67(1H,m, 5"-H), 3.62(1H, m, 4"-H), 3.62(1H, m, (2)"-H), 3.62(1H, m, (5)-H),3.51(1H, t, J=8.9 Hz, 3"-H), 3.28(1H, t, J=7.9 Hz, 2"-H), 1.09(3H, d,J=6.7 Hz, H₃ of (5)'-CH₃), 1.07(1H, d, J=6.7 Hz, H₃ of 5'-CH₃).

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid:D-mannose=2:1:1:2 (L-fucose and D-mannose: each two molecules,unsaturated D-glucuronic acid and D-glucuronic acid: each one molecule).

Sulfate Group:

two molecules (at the 3-position of each L-fucose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (19):##STR11##

Physical properties of the compound (d):

Molecular weight: 1062.

MS m/z: 1061 [M-H⁺ ]⁻.

MS/MS m/z: 97 [HSO₄ ]⁻, 175 [unsaturated hexuronic acid-H⁺ ]⁻, 225[sulfated L-fucose-H₂ O-H⁺ ]⁻, 243 [sulfated L-fucose-H⁺ ]⁻, 405[sulfated L-fucose bonded to D-mannose-H⁺ ]⁻ or [L-fucose bonded tosulfated D-mannose-H⁺ ]⁻, 450 [M-2SO₃ -2H⁺ ]²⁻, 490 [M-SO₃ -2H⁺ ]²⁻.

¹ H-NMR(D₂ O);

δ5.67(1H, d, J=3.7 Hz, (4)"-H), 5.32(1H, br-s, (1)-H), 5.17(1H, d, J=3.5Hz, 1'-H), 5.17(1H, br-s, 1-H), 4.93(1H, d, J=6.4 Hz, (1)"-H), 4.71(1H,m, 1"-H), 4.53(1H, d, J=3.4 Hz, 4'-H), 4.33(1H, q, J=6.7 Hz, 5'-H),4.28(1H, d-d, J=3.5, 11.0 Hz, 2'-H), 4.18(1H, m, H of 5-CH₂), 4.13(1H,m, (2)-H), 4.12(1H, m, (3)"-H), 4.08(1H, m, H of 5-CH₂), 4.07(1H, m,2-H), 3.98(1H, d-d, J=3.4, 11.0, 3'-H), 3.88(1H, m, 5-H), 3.82(1H, m,4"-H), 3.78(1H, m, 3-H), 3.78(1H, m, 4-H), 3.78(1H, m, (3)-H), 3.67(2H,m, H₂ of (5)-CH₂), 3.63(1H, m, (2)"-H), 3.60(1H, m, 3"-H), 3.59(1H, m,5"-H), 3.57(1H, m, (4)-H), 3.57(1H, m, (5)-H), 3.16(1H, t, J=7.9 Hz,2"-H), 1.10(3H, d, J=6.7 Hz, H₃ of 5'-CH₃).

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid:D-mannose=1:1:1:2 (L-fucose, unsaturated D-glucuronic acid andD-glucuronic acid: each one molecule, D-mannose: two molecules).

Sulfate Group:

three molecule (at the 2-and 4-positions of L-fucose and the 6-positionon the reducing end side of D-mannose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (20):##STR12##

Physical properties of the compound (e):

Molecular weight: 1448.

MS m/z: 767 [M+4Na⁺ -6H⁺ ]²⁻, 503.7 [M+3Na⁺ -6H⁺ ]³⁻ and 366.5 [M+Na⁺-5H⁺ ]⁴⁻.

MS/MS m/z: 97 [HSO₄ ]⁻, 225 [sulfated L-fucose-H₂ O-H⁺ ]⁻ 243 [sulfatedL-fucose-H⁺ ]⁻, 345 [disulfated L-fucose) +Na⁺ -2H⁺ ]⁻, 450 [M+3Na⁺-2SO₃ -6H⁺ ]³⁻, 477 [M+3Na⁺ -SO₃ -6H⁺ ]³⁻, 563 [unsaturated D-glucuronicacid and sulfated L-fucose bonded to D-mannose-H⁺ ]⁻ or [unsaturatedD-glucuronic acid and L-fucose bonded to sulfated D-mannose-H⁺ ]⁻, 705[unsaturated D-glucuronic acid and disulfated L-fucose bonded tosulfated D-mannose-H₂ O-H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.58(1H, d, J=3.4 Hz, (4)"-H). 5.35(1H, br-s, (1)-H),5.22(1H, d, J=6.7 Hz, (1)"-H), 5.19(1H, d, J=3.7 Hz, 1'-H), 5.19(1H, d,J=3.7 Hz, (1)'-H), 5.16(1H, d, J=1.8 Hz, 1-H), 4.62(1H, d, J=7.6 Hz,1"-H), 4.50(1H, m, 4'-H), 4.50(1H, m, (4)'-H), 4.30(1H, m, 5'-H),4.30(1H, m, (5)'-H), 4.30(1H, m, H of (5)-CH₂), 4.25(1H, m, 2'-H),4.25(1H, m, (2)-H), 4.25(1H, m, (2)'-H), 4.20(1H, m, H of (5)-CH₂),4.18(1H, m, H of 5-CH₂), 4.16(1H, m, (3)"-H), 4.08(1H, m, H of 5-CH₂),4.07(1H, m, 2-H), 4.02(1H, m, 3'-H), 4.02(1H, m, (3)'-H), 3.85(1H, m,5-H), 3.85(1H, m, (5)-H), 3.78(1H, m, 3-H), 3.78(1H, m, (3)-H), 3.76(1H,m, 4"-H), 3.76(1H, m, 5"-H), 3.75(1H, m, 4-H), 3.75(1H, m, (4)-H),3.58(1H, m, 3"-H), 3.55(1H, m, (2)"-H), 3.18(1H, t, J=8.2 Hz, 2"-H),1.10(3H, d, J=6.7 Hz, H₃ of (5)'-CH₃), 1.09(3H, d, J=6.7 Hz, H₃ of5'-CH₃).

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid:D-mannose=2:1:1:2 (L-fucose and D-mannose: each two molecules,unsaturated D-glucuronic acid and D-glucuronic acid: each one molecule).

Sulfate Group:

six molecules (at the 2- and 4-positions of each L-fucose and the6-position of each D-mannose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (21):##STR13##

Physical properties of the compound (f):

Molecular weight: 644.

MS m/z: 687 [M+2Na⁺ -3H⁺ ]⁻.

MS/MS m/z: 97 [HSO₄ ]⁻, 243 [sulfated L-fucose-H⁺ ]⁻, 421 [unsaturatedD-glucuronic acid bonded to sulfated D-mannose+Na⁺ -H₂ O-2H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.60(1H, d, J=3.4 Hz, 4"-H), 5.24(1H, br-s, 1-H),5.08(1H, d, J=4.0 Hz, 1'-H), 4.94(1H, d, J=6.7 Hz, 1"-H), 4.45(1H, d-d,J=3.1, 10.4 Hz, 3'-H), 4.20(1H, br-s, 2-H), 4.14(2H, m, H₂ of 5-CH₂),4.14(1H, m, 5'-H), 4.09, (1H, m, 3"-H), 4.01(1H, d, J=3.1 Hz, 4'-H),3.91(1H, m, 5-H), 3.85(1H, m, 3-H), 3.85(1H, m, 2'-H), 3.75(1H, t, J=9.8Hz, 4-H), 3.59(1H, t, J=6.7 Hz, 2"-H), 1.06(3H, d, J=6.4 Hz, H₃ of5'-CH₃)

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-mannose=1:1:1 (L-fucose,D-mannose and unsaturated D-glucuronic acid: each one molecule).

Sulfate Group:

two molecules (at the 3-position of L-fucose and the 6-position ofD-mannose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (22):##STR14##

Physical properties of the compound (g):

Molecular weight: 632.

MS m/z: 631 [M-H⁺ ]⁻.

MS/MS m/z: 405 [sulfated L-fucose bonded to D-galactose-H⁺ ]⁻, 551[L-fucose bonded to sulfated L-fucose bonded to D-galactose-H⁺ ]⁻ or[sulfated L-fucose bonded to L-fucose bonded to D-galactose-H⁺ ]⁻ or[M-SO₃ -H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.15(1H, d, J=4.3 Hz, F₁ 1-H), 4.93(1H, d, J=3.7 Hz, F₂1-H), 4.53(1H, d-d, J=2.4, 10.4 Hz, F₁ 3-H), 4.49(1H, d, J=7.6 Hz, G₁1-H), 4.46(1H, d-d, J=3.1, 10.7 Hz, F₂ 3-H), 4.36(1H, q, J=6.7 Hz F₂5-H), 4.14(1H, q, J=6.7 Hz F₁ 5-H), 4.09(1H, d, J=2.4 Hz F₁ 4-H),4.03(1H, d, J=3.1 Hz F₂ 4-H), 3.97(1H, d-d, J=4.3, 10.4 Hz, F₁ 2-H),3.90(1H, br-s, G₁ 4-H, 3.81(1H, d-d, J=3.7, 10.7 Hz, F₂ 2-H), 3.59(1H,m, G₁ 3-H), 3.59(1H, m, G₁ 5-H), 3.59(2H, m, H₂ of G₁ 5-CH₂), 3.56(1H,m, G₁ 2-H), 1.19(3H, d, J=6.7 Hz, H₃ of F₁ 5-CH₃), 1.14(3H, d, J=6.7 Hz,H₃ of F₂ 5-CH₃).

Sugar Composition:

L-fucose: D-galactose=2:1 (two L-fucose molecules and one D-galactosemolecule).

Sulfate Group:

two molecules (at the 3-position of each L-fucose).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (23):##STR15##

Physical properties of the compound (h):

    ______________________________________                                        Molecular weight:                                                                        1358.                                                                MS m/z: 1445 [M+4Na.sup.+ -5H.sup.+ ].sup.-.                                  MS/MS m/z: 97 [HSO.sub.4 ].sup.-, 225 [sulfated L-fucose-                      H.sub.2 O--H.sup.+ ].sup.-, 1197 [M-2SO.sub.3 --H.sup.+ ].sup.-.           .sup.1 H--NMR (D.sub.2 O)                                                     ______________________________________                                         δ5.19(1H, d, J=4.3Hz, F.sub.1 1-H), 4.93(1H, d, J=3.7Hz,                 F.sub.2 1-H), 4.62(1H, m, G.sub.2 1-H), 4.59(1H, m, G.sub.1 1-H),           4.54(1H,                                                                        d-d, J=2.7, 10.6Hz, F.sub.1 3-H), 4.46(1H, m, F.sub.2 3-H), 4.46(1H,         d, J=7.6Hz G.sub.3 1-H), 4.41(1H, br-s, G.sub.1 4-H), 4.41(1H, d,             J=7.6Hz, G.sub.4 1-H), 4.37(1H, q, J=6.4Hz F.sub.2 5-H), 4.27(1H, m,          G.sub.1 3-H), 4.24(1H, br-s, G.sub.3 4-H), 4.21(1H, m, G.sub.3 3-H),         4.19(1H,                                                                        m, G.sub.4 3-H), 4.15(1H, br-s, G.sub.4 4-H), 4.13(1H, q, J=6.7Hz,           F.sub.1 5-H), 4.09(1H, d, J=2.7Hz, F.sub.1 4-H), 4.04(1H, d, J=2.8Hz,         F.sub.2 4-H), 3.98(1H, m, H of G.sub.1 5-CH.sub.2), 3.96(1H, d-d, J=4.3,      10.6Hz, F.sub.1 2-H), 3.88(1H, br-s, G.sub.2 4-H), 3.93(1H, m, H of           G.sub.3 5-CH.sub.2), 3.86(1H, m, G.sub.1 5-H), 3.81(1H, m, F.sub.2 2-H),     3.81(1H,                                                                       m, H of G.sub.1 5-CH.sub.2), 3.80(1H, m, G.sub.3 5-H), 3.80(1H, m, H of       G.sub.3 5-CH.sub.2), 3.66(1H, m, G.sub.2 3-H), 3.65(1H, m, G.sub.1 2-H),     3.64(1H,                                                                       m, H of G.sub.2 5-CH.sub.2), 3.64 (1H, m, H of G.sub.4 5-CH.sub.2) ,         3.61 (1H, m,                                                                    G.sub.4 5-H), 3.58(1H, m, G.sub.2 2-H), 3.56(1H, m, H of G.sub.2            5-CH.sub.2),                                                                    3.56(1H, m, H of G.sub.4 5-CH.sub.2), 3.55(1H, m, G.sub.4 2-H),             3.54(1H,                                                                        m, G.sub.2 5-H), 3.54(1H, m, G.sub.3 2-H), 1.20(3H, d, J=6.7Hz, H.sub.3      of                                                                            F.sub.1 5-CH.sub.3), 1.14(3H, d, J=6.4Hz, H.sub.3  of F.sub.2 5-CH.sub.3)    ______________________________________                                    

Sugar Composition:

L-fucose: D-galactose=1:2 (two L-fucose molecules and four D-galactosemolecules).

Sulfate Group:

five molecules (at the 3-position of each L-fucose, the 3-position ofD-galactose of the reducing end, and the 3-position of D-galactosebonded to the 6-position of D-galactose of the reducing end, and the3-position of D-galactose bonded to the 6-position of theabove-mentioned D-galactose). The peaks in the ¹ H-NMR spectra areassignable respectively to the positions shown by the numerical valuesin the following formula (24): ##STR16##

Physical properties of the compound (i):

Molecular weight: 1288.

MS m/z: 1149 [M+Na⁺ -2SO₃ -2H⁺ ]⁻.

MS/MS m/z: 97 [HSO₄ ]⁻, 345 [disulfated L-fucose+Na⁺ -2H⁺ ]⁻.

¹ H-NMR(D₂ O); δ5.68(1H, d, J=3.4 Hz, (4)"-H), 5.34(1H, br-s, (1)-H),5.19(1H, m, 1-H), 5.19(1H, m, (1)'-H), 4.94(1H, m, 1'-H), 4.94(1H, d,J=6.4 Hz, (1)"-H), 4.72(1H, d, J=7.9 Hz, 1"-H), 4.54(1H, m, (4)'-H),4.48(1H, d-d, J=3.3, 10.6 Hz, 3'-H), 4.38(1H, q, J=6.4 Hz, 5'-H),4.34(1H, q, J=6.7 Hz, (5)'-H), 4.29(1H, m, (2)'-H), 4.20(1H, m, H of5-CH₂), 4.14(1H, m, (2)-H), 4.13(1H, m, (3)"-H), 4.09(1H, m, H of5-CH₂), 4.08(1H, m, 2-H), 4.05(1H, d, J=3.3 Hz, 4'-H), 3.99(1H, m,(3)'-H), 3.89(1H, m, 5-H), 3.83(1H, m, 4"-H), 3.81(1H, m, 2'-H),3.80(1H, m, 4-H), 3.78(1H, m, 3-H), 3.78(1H, m, (3)-H), 3.68(2H, m, H₂of (5)-CH₂), 3.62(1H, m, (2)"-H), 3.60(1H, m, 3"-H), 3.60(1H, m, 5"-H),3.58(1H, m, (5)-H), 3.56(1H, m, (4)-H), 3.17(1H, t, J=7.9 Hz, 2"-H),1.15(3H, d, J=6.4 Hz, H₃ of 5'-CH₃), 1.11(3H, d, J=6.7 Hz, H₃ of(5)'-CH₃).

Sugar Composition:

L-fucose: unsaturated D-glucuronic acid: D-glucuronic acid:D-mannose=2:1:1:2 (L-fucose and D-mannose: each two molecules,unsaturated D-glucuronic and D-glucuronic acid: each one molecule).

Sulfate Group:

four molecules (at the 3-position of L-fucose bonded to D-mannose of thereducing end, the 2- and 4-positions of another L-fucose, and the6-position of D-mannose on the reducing end side).

The peaks in the ¹ H-NMR spectra are assignable respectively to thepositions shown by the numerical values in the following formula (25):##STR17##

Also, the sugar compounds of the first invention of the presentinvention can be produced by treating fucoidan with a cell extract orculture supernatant of the bacterium of the third invention of thepresent invention belonging to the genus Fucoidanobacter.

The strain of the third invention of the present invention may be anarbitrary one so long as it is a strain of the present inventionbelonging to the genus Fucoidanobacter. As a particular example thereof,citation may be made of Fucoidanobacter marinus SI-0098 strain.

This strain Fucoidanobacter marinus SI-0098, which has been found outfor the first time by the present inventors from seawater in Aomori, hasthe following mycological properties.

2. Fucoidanobacter marinus SI-0098 Strain

a. Morphological Properties

(1) Short rod (sometimes diplococcal);

width: 0.5-0.7 μm

length: 0.5-0.7 μm

(2) Spore: none

(3) Gram-staining: -

b. Physiological properties

(1) Growth temperature range: capable of growing at 37° C., appropriategrowth temperature ranging from 15 to 28° C.

(2) Attitude to oxygen: aerobic

(3) Catalase: +

(4) Oxidase: -

(5) Urease: -

(6) Hydrolysis starch: +

gelatin: -

casein: -

esculin: +

(7) Reduction of nitrate: -

(8) Indole formation: -

(9) Hydrogen sulfide formation: +

(10) Solidification of milk: -

(11) Sodium requirement: +

(12) Salt requirement

Growth in NaCl-free medium: -

Growth in 1% NaCl medium: -

Growth in seawater medium: +

(13) Quinone: menaquinone 7

(14) GC content of intracellular DNA: 61%

(15) Diaminopimelic acid in cell wall: -

(16) Glycolyl test: -

(17) Presence of hydroxy fatty acid: +

(18) OF-test: O

(19) Colony color: forming no characteristic color

(20) Motility: yes

(21) Gliding: none

(22) Flagellum: polar monotrichous.

According to the classification described in Bergey's Manual ofDeterminative Bacteriology, 9 (1994), this strain falls in Group 4(Gram-negative aerobic bacilli and cocci). However, this strain largelydiffers from the bacteria belonging to Group 4 in having menaquinone inthe electron transport chain and containing 61% of GC. Fundamentally,gram-negative bacteria have ubiquinone in the electron transport chainwhile gram-positive bacteria have menaquinone.

Although gram-negative bacteria belonging to the genera Flavobacteriumand Cytophaga exceptionally have menaquinone in the electron transportchain, they are largely different in GC content from the above-mentionedstrain, such that Cytophaga arvensicola, which is a soil bacteriumcontains from 43 to 46% of GC and Cytophaga diffluens, C. fermentans, C.marina and C. uliginosa, which are marine bacteria each contains 42% ofGC. When this strain is compared in the homology in 16SrDNA sequencewith strains which have been identified, even the most homologous one(Verrucomicrobium spinosum) shows a homology of 76.6% therewith. It iswidely known that two bacteria with a homology of 90% or less with eachother are different in genus. Accordingly, the present inventors havedecided that this strain is a novel bacterium belonging to none of theexisting genera and named it Fucoidanobacter marinus SI-0098.

The above strain is designated as Fucoidanobacter marinus SI-0098 andhas been deposited at National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (1-3,Higashi 1-chome, Tsukuba, Ibaragi, 305 JAPAN) under the accession numberFERM BP-5403 (original deposition was made on Mar. 29, 1995 and transferto international deposition was requested on Feb. 15, 1996).

It is possible that a microorganism of the third invention of thepresent invention belonging to the genus Fucoidanobacter is incubated ina medium and fucoidan is treated with the cell extract or the culturesupernatant to thereby liberate the sugar compounds of the firstinvention of the present invention. The microorganism to be usedtherefor may be an arbitrary one, so long as it belongs to the genusFucoidanobacter and is capable of giving the sugar compounds of thefirst invention of the present invention when fucoidan is treated withthe cell extract or the culture supernatant thereof. As a particularexample of the strain, citation may be made of Fucoidanobacter marinusSI-0098 strain.

The nutrients to be added to the medium of the bacterial strainbelonging to the genus Fucoidanobacter may be arbitrary ones so long asthe strain employed can utilize them so as to give the cell extract orthe culture supernatant by which the sugar compounds of the firstinvention of the present invention can be produced. Appropriate examplesof the carbon source include fucoidan, marine alga powder, alginic acid,fucose, glucose, mannitol, glycerol, saccharose, maltose, lactose andstarch, while appropriate examples of the nitrogen source include yeastextract, peptone, casamino acids, corn steep liquor, meat extract,defatted soybean, ammonium sulfate and ammonium chloride. The medium mayfurther contain inorganic substances and metal salts such as sodiumsalts, phosphates, potassium salts, magnesium salts and zinc salts.

This strain also grows well in seawater or artificial seawatercontaining the above-mentioned nutritional sources.

To incubate the bacterium belonging to the genus of Fucoidanobacteraccording to the third invention of the present invention, it isgenerally preferable that the incubation temperature ranges from 15 to30° C. and the pH value of the medium ranges from 5 to 9. Theendo-fucoidan-lyase activity in the cell extract and culture supernatantattains the maximum by incubating the strain under aeration andagitation for 5 to 72 hours. As a matter of course, the incubationconditions are appropriately selected depending on the strain employed,the medium composition, etc. so as to achieve the maximum activity ofendo-fucoidan-lyase of the intracellular extract and the culturesupernatant.

The Fucoidanobacter marinus SI-0098 strain is incubated in anappropriate medium and, after the completion of the incubation, theculture medium is centrifuged to thereby give the cells and the culturesupernatant. Further the cells thus harvested are disrupted by a meanscommonly employed for disrupting cells such as ultrasonication. Bycentrifuging the disrupted cells, a cell extract can be obtained.

Subsequently, the culture supernatant or the cell extract isconcentrated by ultrafiltration and mixed with a phosphate buffercontaining sodium chloride. Then fucoidan is added thereto and reactedtherewith.

After the completion of the reaction, the reaction mixture isfractionated by using a column for molecular weight fractionation. Thusthe sugar compounds of the first invention of the present invention canbe obtained.

The sugar compounds of the first invention of the present invention areuseful as a reagent for the sugar chain technology. Bypyridyl-(2)-aminating (PA) these compounds in accordance with the methoddescribed in Japanese Patent Publication No. 65108/1993, it is possibleto give PA compounds which are highly useful as a reagent for the sugarchain technology. Furthermore, it is expected that the physiologicalactivities of the sugar compounds of the present invention make themapplicable to anticancer, cancerous metastasis-inhibitory and antiviraldrugs.

Best Mode for Carrying Out the Invention

The following Examples will be given in order to illustrate examples ofthe process for producing the sugar compounds of the first invention ofthe present invention, though it is to be understood that the presentinvention is not restricted thereto.

EXAMPLE 1

Flavobacterium sp. SA-0082 (FERM BP-5402) was inoculated into 600 ml ofa medium comprising an artificial seawater (pH 7.5, mfd. by JamarinLaboratory) containing 0.1% of glucose, 1.0% of peptone and 0.05% ofyeast extract which had been pipetted into a 2-1 Erlenmeyer flask andsterilized at 120° C. for 20 minutes.

Then the strain was incubated therein at 24° C. for 20 hours to therebygive a seed culture. Into a 30-1 jar fermenter was fed 20 l of a mediumcomprising an artificial seawater (pH 7.5, mfd. by Jamarin Laboratory)containing 0.3% of fucoidan originating in Kjellmaniella crassifolia,0.5% of peptone, 0.01% of yeast extract and 0.01% of a defoaming agent(KM70 mfd. by Shin-Etsu Chemical Co., Ltd.) and sterilized at 120° C.for 20 minutes. After cooling, the medium was inoculated with 600 ml ofthe above-mentioned seed culture, which was then incubated therein at24° C. for 20 hours under aerating at a rate of 10 l/min and agitatingat 125 rpm. After the completion of the incubation, the culture mediumwas centrifuged to thereby obtain the cells and the culture supernatant.

The culture supernatant was concentrated by ultrafiltration (exclusionmolecular weight of membrane: 10,000, mfd. by Amicon) and theendo-fucoidan-lyase of the present invention was assayed. Thus anactivity of 6 mU/ml of the culture medium was detected.

Separately, the cells obtained by the main incubation were suspended ina 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM of sodiumchloride, disrupted by ultrasonication and centrifuged to thereby give acell extract. When the endo-fucoidan-lyase of the present invention inthis cell extract was assayed, an activity of 20 mU/ml of the culturemedium was detected.

To the above-mentioned concentrate of the culture supernatant was addedammonium sulfate so as to establish 90% saturation finally. Afterdissolving by stirring, the mixture was centrifuged and the precipitatewas suspended in the same buffer as the above-mentioned one in which thecells were suspended. Then the suspension was thoroughly dialyzedagainst a 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM ofsodium chloride. After eliminating the precipitate formed by thedialysis by centrifugation, it was adsorbed on a DEAE-Sepharose FFcolumn which had been equilibrated with a 20 mM acetate-phosphate buffer(pH 7.5) containing 50 mM of sodium chloride. Then the adsorbed matterwas well washed with the same buffer and developed by linear gradientelution with sodium chloride of 50 mM to 600 mM.

The active fractions were combined and sodium chloride was added theretoso as to give a final concentration of 4 M. Next, it was adsorbed on aPhenyl Sepharose CL-4B column which had been equilibrated with a 20 mMphosphate buffer (pH 8.0) containing 4 M of sodium chloride. Then theadsorbed matter was well washed with the same buffer and developed bylinear gradient elution with sodium chloride of 4 M to 1 M. The activefractions were combined and concentrated with an ultrafilter (mfd. byAmicon). Next, it was subjected to gel filtration with the use ofSephacryl S-200 gel which had been equilibrated with a 10 mM phosphatebuffer containing 50 mM of sodium chloride. The active fractions werecombined and sodium chloride was added thereto so as to give a finalconcentration of 3.5 M. Next, it was adsorbed on a Phenyl Sepharose HPcolumn which had been equilibrated with a 10 mM phosphate buffer (pH 8)containing 3.5 M of sodium chloride. Then the adsorbed matter was washedwith the same buffer and developed by linear gradient elution withsodium chloride of 3.5 M to 1.5 M. The active fractions were combined tothereby give the purified enzyme. The molecular weight of the enzymedetermined from the retention time in Sephacryl S-200 was about 70,000.Table 1 summarizes the above-mentioned purification steps.

                  TABLE 1                                                         ______________________________________                                                       Total   Total   Specific                                                                               protein activity activity                                                    Step (mg) (mU) (mU/mg) Yield           ______________________________________                                                                              (%)                                     cell extract   980     114,000   116  100                                       ammonium sulfate-salting out 473 108,000   228 94.7                           DEAE-Sepharose FF 216  86,400   400 75.8                                      Phenyl Sepharose CL-4B 21.9  57,300  2,620 50.3                               Sephacryl S-200 3.70  46,200 12,500 40.5                                      Phenyl Sepharose HP 1.53  41,200 27,000 36.1                                ______________________________________                                    

EXAMPLE 2

Flavobacterium sp. SA-0082 (FERM BP-5402) was inoculated into 600 ml ofa medium comprising an artificial seawater (pH 7.5, mfd. by JamarinLaboratory) containing 0.25% of glucose, 1.0% of peptone and 0.05% ofyeast extract which had been pipetted into a 2-1 Erlenmeyer flask andsterilized at 120° C. for 20 minutes.

Then the strain was incubated therein at 24° C. for 24 hours to therebygive a seed culture. Into a 30-1 jar fermenter was fed 20 l of a mediumcomprising an artificial seawater (pH 7.5, mfd. by Jamarin Laboratory)containing 0.25% of glucose, 1.0% of peptone, 0.05% of yeast extract and0.01% of a defoaming agent (KM70 mfd. by Shin-Etsu Chemical Co., Ltd.)and sterilized at 120° C. for 20 minutes. After cooling, the medium wasinoculated with 600 ml of the above-mentioned seed culture, which wasthen incubated therein at 24° C. for 24 hours under aerating at a rateof 10 l/min and agitating at 125 rpm. After the completion of theincubation, the culture medium was centrifuged to thereby give the cellsand the culture supernatant.

The culture supernatant was concentrated by ultrafiltration (exclusionmolecular weight of membrane: 10,000, mfd. by Amicon) and theendo-fucoidan-lyase of the present invention was assayed. Thus anactivity of 1 mU/ml of the culture medium was detected.

Separately, the cells obtained by the main incubation were suspended ina 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM of sodiumchloride, disrupted by ultrasonication and centrifuged to thereby give acell extract. When the endo-fucoidan-lyase of the present invention inthis cell extract was assayed, an activity of 5 mU/ml of the culturemedium was detected.

To this extract was added ammonium sulfate so as to establish 90%saturation finally. After dissolving by stirring, the mixture wascentrifuged and the precipitate was suspended in the same buffer as theabove-mentioned one in which the cells were suspended. Then thesuspension was thoroughly dialyzed against a 20 mM acetate-phosphatebuffer (pH 7.5) containing 50 mM of sodium chloride. After eliminatingthe precipitate formed by the dialysis by centrifugation, it wasadsorbed on a DEAE-Sepharose FF column which had been equilibrated witha 20 mM acetate-phosphate buffer (pH 7.5) containing 50 mM of sodiumchloride. Then the adsorbed matter was well washed with the same bufferand developed by linear gradient elution with sodium chloride of 50 mMto 600 mM. The active fractions were combined and sodium chloride wasadded thereto so as to give a final concentration of 4 M. Next, it wasadsorbed on a Phenyl Sepharose CL-4B column which had been equilibratedwith a 20 mM phosphate buffer (pH 8.0) containing 4 M of sodiumchloride. Then the adsorbed matter was well washed with the same bufferand developed by linear gradient elution with sodium chloride of 4 M to1 M. The active fractions were combined and concentrated with anultrafilter. Next, it was subjected to gel filtration with the use ofSephacryl S-300 which had been equilibrated with a 10 mM phosphatebuffer containing 50 mM of sodium chloride. The active fractions werecombined. The molecular weight of the enzyme determined from theretention time in Sephacryl S-300 was about 460,000. Next, the activefraction was dialyzed against a 10 mM phosphate buffer (pH 7) containing250 mM of sodium chloride. The enzyme solution was adsorbed on a Mono QHR5/5 column which had been equilibrated with a 10 mM phosphate buffer(pH 7) containing 250 mM of sodium chloride. The adsorbed matter waswell washed with the same buffer and developed by linear gradientelution with sodium chloride of 250 mM to 450 mM. The active fractionswere combined to thereby give the purified enzyme. Table 2 summarizesthe above-mentioned purification steps.

                  TABLE 2                                                         ______________________________________                                                         Total   Total   Specific                                        protein activity activity Yield                                              Step (mg) (mU) (mU/mg) (%)                                                  ______________________________________                                        cell extract     61,900  101,000 1.63   100                                     ammonium sulfate-salting out 33,800  88,600 2.62 87.7                         DEAE-Sepharose FF  2,190  40,400 18.4 40.O                                    Phenyl Sepharose CL-4B 48.2  29,000 601 28.7                                  Sephacryl S-300 7.24  19,600  2,710 19.4                                      Mono Q 0.824  15,000 18,200 14.9                                            ______________________________________                                    

EXAMPLE 3

Purified fucoidan originating in Kjellmaniella crassifolia was treatedwith the endo-fucoidan-lyase of the present invention obtained inExample 1 (the intracellular enzyme) to thereby prepare the degradationproducts thereof.

Namely, 16 ml of a 2.5% fucoidan solution, 12 ml of a 50 mM phosphatebuffer (pH 7.5), 4 ml of a 4 M solution of sodium chloride and 8 ml of a32 mU/ml solution of the endo-fucoidan-lyase of the present inventionwere mixed together and reacted at 25° C. for 48 hours.

Then the reaction mixture was subjected to molecular weightfractionation by using a Cellulofine GCL-300 column (mfd. by SeikagakuKogyo) and the fraction of molecular weight of not more than 2,000 wastaken up. After desalting with a Micro Acilyzer G3 (mfd. by AsahiChemical Industry Co., Ltd.), this fraction was separated into threefractions by DEAE-Sepharose FF to thereby give 41 mg, 69 mg and 9.6 mgof the above-mentioned compounds (a), (b) and (c), respectively.

EXAMPLE 4

Purified fucoidan originating in Kjellmaniella crassifolia was treatedwith the endo-fucoidan-lyase of the present invention obtained inExample 2 (the extracellular enzyme) to thereby prepare the degradationproducts thereof.

Namely, 16 ml of a 2.5% fucoidan solution, 12 ml of a 50 mM phosphatebuffer (pH 7.5), 4 ml of a 4 M solution of sodium chloride and 8 ml of a32 mU/ml solution of the endo-fucoidan-lyase of the present inventionwere mixed together and reacted at 25° C. for 48 hours.

Then the reaction mixture was subjected to molecular weightfractionation by using a Cellulofine GCL-300 column (mfd. by SeikagakuKogyo) and the fraction of molecular weight of not more than 2,000 wastaken up.

After desalting with a Micro Acilyzer G3 (mfd. by Asahi ChemicalIndustry Co., Ltd.), this fraction was separated into three fractions byDEAE-Sepharose FF and freeze-dried to thereby give 40 mg, 65 mg and 9.2mg of the above-mentioned compounds (a), (b) and (c), respectively.

EXAMPLE 5

Fucoidanobacter marinus SI-0098 strain (FERM BP-5403) was inoculatedinto 600 ml of a medium comprising an artificial seawater (pH 7.5, mfd.by Jamarin Laboratory) containing 0.3% of fucoidan originating inKjellmaniella crassifolia, 0.5% of peptone, 0.05% of yeast extract and0.01% of a defoaming agent (KM70 mfd. by Shin-Etsu Chemical Co., Ltd.)and sterilized (120° C., 20 minutes). Then the strain which had beenpipetted into a 2-1 Erlenmeyer flask and sterilized at 120° C. for 20minutes. Then the strain was incubated therein at 25° C. for 48 hoursunder agitating at 120 rpm. After the completion of the incubation, theculture medium was centrifuged to thereby give the cells and the culturesupernatant.

The culture supernatant was concentrated by ultrafiltration (exclusionmolecular weight of membrane: 10,000, mfd. by Amicon) and theendo-fucoidan-lyase of the present invention was assayed. Thus anactivity of 0.2 mU/ml of the culture medium was detected.

Separately, the cells obtained by the main incubation were suspended ina 20 mM acetate-phosphate buffer (pH 7.5) containing 200 mM of sodiumchloride, disrupted by ultrasonication and centrifuged to thereby give acell extract. When the endo-fucoidan-lyase of the present invention inthis cell extract was assayed, an activity of 20 mU/ml of the culturemedium was detected.

EXAMPLE 6

Purified fucoidan originating in Kjellmaniella crassifolia was treatedwith the intracellular enzyme of the Fucoidanobacter marinus SI-0098strain of the present invention obtained in Example 5 to thereby preparethe degradation products thereof.

Namely, 16 ml of a 2.5% fucoidan solution, 20 ml of a 100 mM phosphatebuffer (pH 8.0) containing 800 mM of sodium chloride and 4 ml of a 20mU/ml solution of the intracellular enzyme of the Fucoidanobactermarinus SI-0098 strain of the present invention obtained in Example 5were mixed together and reacted at 25° C. for 48 hours.

Then the reaction mixture was subjected to molecular weightfractionation by using a Cellulofine GCL-300 column and the fraction ofmolecular weight of not more than 2,000 was taken up. After desaltingwith a Micro Acilyzer G3, this fraction was separated into threefractions by DEAE-Sepharose FF and freeze-dried to thereby give 38 mg,60 mg and 8.2 mg of the above-mentioned compounds (a), (b) and (c),respectively.

The present invention thus provides a novel endo-fucoidan-lyase which isuseful in the analysis of the structure of fucoidan, the identificationof the enzymatic degradation products of fucoidan and studies relatingto fucoidan, for example, sugar compounds usable in the detection ofbiological activities of fucoidan, the partial degradation of fucoidanand the production of fucoidan oligosaccharides.

What is claimed is:
 1. An oligosaccharide compound represented by thefollowing general formula (1) or (2), wherein at least one alcoholichydroxyl group has been sulfated, or its salt: ##STR18## Y representshydrogen or a group represented by the following formula (4) or (5):##STR19## provided that X and Y are not hydrogen at the same time; and Zrepresents hydrogen or a group represented by the following formula (6):##STR20##
 2. An oligosaccharide compound represented by the followingformula (7) or its salt:
 3. An oligosaccharide compound represented bythe following formula (8) or its salt:
 4. An oligosaccharide compoundrepresented by the following formula (9) or its salt:
 5. Anoligosaccharide compound represented by the following formula (10) orits salt:
 6. An oligosaccharide compound represented by the followingformula (11) or its salt:
 7. An oligosaccharide compound represented bythe following formula (12) or its salt:
 8. An oligosaccharide compoundrepresented by the following formula (13) or its salt:
 9. Anoligosaccharide compound represented by the following formula (14) orits salt:
 10. An oligosaccharide compound represented by the followingformula (15) or its salt: